Carcinoma associated antigen (SK1) monoclonal antibodies against SK1, methods of producing these antibodies and use therefor

ABSTRACT

Carcinoma associated antigen (SK1) and monoclonal antibodies and methods for detecting and ameliorating malignant disease. The monoclonal antibodies are specifically reactive with epitopes present on SK1.

[0001] This application is a continuation-in-part of application U.S.Ser. No. 07/827,717, filed Jan. 29, 1992.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] This invention relates to a novel carcinoma associated antigen(SK1) which is associated with various malignancies and monoclonalantibodies specific for epitopes on SK1.

[0004] 2. Description of the Background Art

[0005] Murine monoclonal antibodies have been shown to mediate effectivecytotoxicity to target cells in vitro; however, when utilized in vivowith humans, they have not achieved remarkable results. This ispartially due to (i) the foreign nature of the injected murine proteinswhich leads to the development of a human anti-mouse antibody (HAMA)response, and (ii) the human effector functions which may not be fullyactivated by a murine antibody. In contrast, the dramatic effects whichhave been observed in systemically treating septic patients withpurified human monoclonal or polyclonal antibodies (Ziegler, et al., TheNew England J.Med, 324:429-436, 1991; Kurtzberg, et al.,Am.J.Pediatr.Hematol.Oncol., 9:299-301, 1987) and by intralesionaltherapy of melanoma suggests that the clinical use of human monoclonalantibodies (HuMAbs) (Irie, et al., Proc.Natl.Acad.Sci. USA,83:8694-8698, 1986) will be successful. Thus, the potential use of humanMabs for cancer therapy is attractive.

[0006] Recent progress in the field of HuMAb technology has made itpossible to generate numerous hybridomas of various specificities.Combined with knowledge gained in the understanding of the human immuneresponse to cancer antigens (Lloyd, et al., Cancer Res., 49:3445-3451,1989), several HuMAbs against tumor associated antigens (TAAs) have beenproduced and characterized. The reported tumor associated antigensrecognized by HuMAbs include cell surface (Yoshikawa, et al., Jpn. J.Cancer Res. (Gann), 80:546-553, 1989; Yamaguchi, et al.,Proc.Natl.Acad.Sci.USA, 84:2416-2420; Haspel, et al., Cancer Res.,45:3951-3961, 1985; Cote, et al., Proc.Natl.Acad.Sci.USA,83:2959-2963,1986; Glassy, Cancer Res., 47:5181-5188, 1987; andBorup-Christensen, et al., Cancer Detect. Prevent.Suppl., 1:207-215),cytoplasmic (Haspel, et al., Cancer Res., 45:3951-3961, 1985; Cote, etal., Proc.Natl.Acad.Sci.USA, 83:2959-2963, 1986; Glassy, Cancer Res.,47:5181-5188; Borup-Christensen, et al., Cancer Detect Prevent Suppl.,1:207-215,1987; Kan-Mitchell, et al., Cancer Res., 49:4536-4541, 1989;and Yoshikawa, et al., Jpn.J. Cancer Res., 77:1122-1133, 1986), andnuclear antigens (McKnight, et al., Hum.Antibod. Hybridomas, 1:125-129,1990).

[0007] At present, methods of limited effectiveness exist for treatmentof various malignancies. Those drugs which are administered generallyhave severe side effects associated with their use. Accordingly, thereexists a significant need to identify and purify an antigen associatedwith malignant diseases and to produce monoclonal antibodies which bindto epitopes on this antigen. Further, these antibodies are suitableagents for the diagnosis and treatment of malignancies expressing theSK1 antigen.

SUMMARY OF THE INVENTION

[0008] One way to ameliorate malignancies would be to suppress cellswhich preferentially express an antigen associated with carcinoma. Thissuppression could be accomplished, for example, by active immunizationusing an antigen, or a derivative thereof, preferentially present inmalignant cells or by passive immunization by providing antibody to theantigen.

[0009] In order to provide a means to ameliorate malignant disease theinvention provides substantially purified antigen which ispreferentially expressed by malignant cells and monoclonal antibodieswhich bind to epitopes on the antigen. These monoclonal antibodies, ifdesired, can be labeled for therapeutic or diagnostic use.

[0010] An object of the present invention is to provide a method ofdetecting the carcinoma associated antigen (SK1) preferentiallyexpressed in various malignant cells and tissues using a detectablylabeled monoclonal antibody which binds to SK1 and determining whetherthe detectably labeled monoclonal antibody has bound to SK1.

[0011] Another object of the present invention is to provide methods forthe in vitro and in vivo diagnosis of malignancy using detectablylabeled monoclonal antibodies which react with an epitope present onSK1.

[0012] Another object of the invention is to provide methods forameliorating malignant disease in an animal using unlabeled ortherapeutically labeled monoclonal antibodies which react with SK1.

[0013] Alternatively, the invention provides methods for amelioratingmalignant disease in an animal by inducing an immune response to themalignancy by immunizing the animal with SK1.

[0014] The present invention thus relates to a method of detecting SK1which comprises contacting a source suspected of containing SK1 with adiagnostically effective amount of detectably labeled monoclonalantibody, or fragment thereof, having the specificity of a monoclonalantibody of the invention and determining whether the antibody binds tothe source.

[0015] The invention further relates to a method of suppressingmalignant disease in an animal which comprises administering to theanimal a therapeutically effective amount of a (1) monoclonal antibody,or fragment thereof, wherein the antibody has the specificity of amonoclonal antibody of the invention, or (2) SK1.

[0016] A major advantage in the therapeutic and diagnostic use of SK1and monoclonal antibodies which bind to epitopes of SK1 is that the SK1antigen occurs at high frequency in malignant cells. Consequently, thereis a much greater probability of binding occurring to a malignant cellthan to a normal cell. As a result of this fact, it is possible to useconcentrations of the monoclonal antibody of the invention which areclinically effective, but pose minimal or no risk to normal host cells.

BRIEF DESCRIPTION OF THE DRAWINGS

[0017]FIG. 1 shows tumor size versus time in an SK1 nude mouse xenograftmodel. (=PBS control; ▪=control IgM; and ▴=HuMAb to antigen SK1).

[0018]FIG. 2 is an SDS-PAGE gel showing the 42-46 kD SK1 antigen inhuman cell lines (S=supernatant; P=nuclear pellet).

DETAILED DESCRIPTION OF THE INVENTION

[0019] The present invention relates to a substantially purified antigen(SK1) which is preferentially expressed by malignant cells and tomonoclonal antibodies with epitopic specificity for SK1. Thesemonoclonal antibodies are highly useful for both the in vitro and invivo immunological detection of antigens associated with thesemalignancies and for immunotherapy of cells bearing SK1.

[0020] In a preferred embodiment of the invention a monoclonal antibodyis disclosed which binds to an epitope on SK1. This specificity enablesthe monoclonal antibody, and like monoclonal antibodies with likespecificity, to be used to suppress growth of malignant cells havingSK1. As a consequence, these monoclonal antibodies are useful inameliorating malignant diseases such as colorectal carcinoma, gastriccancer, pancreatic cancer, and adenocarcinoma.

Methods of Producing and Characterizing Monoclonal Antibodies to SK1

[0021] The general method used for production of hybridomas secretingmonoclonal antibodies is well known (Kohler and Milstein, Nature,256:495, 1975). Briefly, lymph nodes from a patient with colon cancerwere harvested from colectomy specimens and lymphocytes isolated. Thelymphocytes were then immunized in vitro with carcinoembryonic antigen.Hybridomas were screened for production of antibody which bound to acolon cancer cell line.

[0022] In one aspect, the present invention is directed to monoclonalantibodies, and hybridomas which produce them, which are reactive withSK1. The isolation of hybridomas secreting monoclonal antibodies withthe reactivity of the monoclonal antibodies of the invention can beaccomplished using routine screening techniques to determine theelementary reaction pattern of the monoclonal antibody of interest.Thus, if a monoclonal antibody being tested binds with SK1, then theantibody being tested and the antibody produced by the hybridomas of theinvention are equivalent.

[0023] Alternatively, since the invention teaches the substantialpurification of the novel SK1 antigen, it is now possible to use thisantigen for purposes of immunization to produce more hybridomas whichsecrete monoclonal antibodies specific for the SK1 antigen. Thisapproach would have the added advantage of decreasing the repertoire ofmonoclonal antibodies generated by limiting the number of antigenicdeterminants presented at immunization. The monoclonal antibodies soproduced could be screened for specificity for SK1 using standardtechniques, for example, by binding SK1 to microtiter plate andmeasuring binding of the monoclonal antibody by an ELISA assay. The term“substantially pure form” when applied to SK1 means that SK1 isessentially free of other proteins with which SK1 is normally associatedin nature.

[0024] It is also possible to evaluate, without undue experimentation, amonoclonal antibody to determine whether it has the same specificity asa monoclonal antibody of the invention by determining whether themonoclonal antibody being tested prevents a monoclonal antibody of theinvention from binding 1) to the SK1 antigen, or 2) a malignant cellexpressing the SK1 antigen with which the monoclonal antibody of theinvention is normally reactive. If the monoclonal antibody being testedcompetes with the monoclonal antibody of the invention, as shown by adecrease in binding by the monoclonal antibody of the invention, then itis likely that the two monoclonal antibodies bind to the same, or aclosely related, epitope.

[0025] Still another way to determine whether a monoclonal antibody hasthe specificity of a monoclonal antibody of the invention is topre-incubate the monoclonal antibody of the invention with the SK1antigen with which it is normally reactive, and determine if themonoclonal antibody being tested is inhibited in its ability to bind theantigen. If the monoclonal antibody being tested is inhibited then, inall likelihood, it has the same, or a closely related, epitopicspecificity as the monoclonal antibody of the invention.

[0026] While the in vivo use of a monoclonal antibody from a foreigndonor species in a different host recipient species is usuallyuncomplicated, a potential problem which may arise is the appearance ofan adverse immunological response by the host to antigenic determinantspresent on the donor antibody. In some instances, this adverse responsecan be so severe as to curtail the in vivo use of the donor antibody inthe host. Further, the adverse host response may serve to hinder themalignancy suppressing efficacy of the donor antibody. One way in whichit is possible to circumvent the likelihood of an adverse immuneresponse occurring in the host is by using chimeric antibodies (Sun, etal., Hybridoma, 5 (Supplement 1):S17, 1986; Oi, et al., Bio Techniques,4(3): 214, 1986). Chimeric antibodies are antibodies in which thevarious domains of the antibodies' heavy and light chains are coded forby DNA from more than one species. Typically, a chimeric antibody willcomprise the variable domains of the heavy (V_(H)) and light (V_(L))chains derived from the donor species producing the antibody of desiredantigenic specificity, and the constant domains of the heavy (C_(H)) andlight (C_(L)) chains derived from the host recipient species. It isbelieved that by reducing the exposure of the host immune system to theantigenic determinants of the donor antibody domains, especially thosein the C_(H) region, the possibility of an adverse immunologicalresponse occurring in the recipient species will be reduced. Thus, forexample, it is possible to produce a chimeric antibody for in vivoclinical use in humans which comprises mouse V_(H) and V_(L) domainscoded for by DNA isolated from a hybridoma of the invention, such asATCC HB 10905, and C_(H) and C_(L) domains coded for with DNA isolatedfrom a human leukocyte.

[0027] Although the present invention encompasses all monoclonalantibodies which recognize the novel carcinoma antigen SK1, especiallypreferred are monoclonal antibodies of human origin. Such humanmonoclonal antibodies are exemplified by HuMAb(SK1) which is an IgMantibody produced by a hybridoma having accession number ATCC HB 10905.

[0028] Under certain circumstances, monoclonal antibodies of one isotypemight be more preferable than those of another in terms of theirdiagnostic or therapeutic efficacy. For example, from studies onantibody-mediated cytolysis, it is known that unmodified mousemonoclonal antibodies of isotype gamma-2a and gamma-3 are generally moreeffective in lysing target cells than are antibodies of the gamma-1isotype. This differential efficacy is thought to be due to the abilityof the gamma-2a and gamma-3 isotypes to more actively participate in thecytolytic destruction of target cells. Particular isotypes of amonoclonal antibody can be prepared either directly, by selecting fromthe initial fusion, or prepared secondarily, from a parental hybridomasecreting a monoclonal antibody of different isotype by using the sibselection technique to isolate class-switch variants (Steplewski, etal., Proceedings of the National Academy of Science, U.S.A., 82:8653,1985; Spira, et al., Journal of Immunological Methods, 74:307, 1984).Thus, the monoclonal antibodies of the invention would includeclass-switch variants having specificity for an epitope on SK1.

[0029] The isolation of other hybridomas secreting monoclonal antibodieswith the specificity of the monoclonal antibodies of the invention canalso be accomplished by one of ordinary skill in the art by producinganti-idiotypic antibodies (Herlyn, et al., Science, 232:100, 1986). Ananti-idiotypic antibody is an antibody which recognizes uniquedeterminants present on the monoclonal antibody produced by thehybridoma of interest. These determinants are located in thehypervariable region of the antibody. It is this region which binds to agiven epitope and, thus, it is responsible for the specificity of theantibody. The anti-idiotypic antibody can be prepared by immunizing ananimal with the monoclonal antibody of interest. The animal immunizedwill recognize and respond to the idiotypic determinants of theimmunizing antibody by producing an antibody to these idiotypicdeterminants. By using the anti-idiotypic antibodies of the secondanimal, which are specific for the monoclonal antibodies produced by asingle hybridoma which was used to immunize the second animal, it is nowpossible to identify other clones with the same idiotype as the antibodyof the hybridoma used for immunization.

[0030] Idiotypic identity between monoclonal antibodies of twohybridomas demonstrates that the two monoclonal antibodies are the samewith respect to their recognition of the same epitopic determinant.Thus, by using antibodies to the epitopic determinants on a monoclonalantibody it is possible to identify other hybridomas expressingmonoclonal antibodies of the same epitopic specificity.

[0031] It is also possible to use the anti-idiotype technology toproduce monoclonal antibodies which mimic an epitope. For example, ananti-idiotypic monoclonal antibody made to a first monoclonal antibodywill have a binding domain in the hypervariable region which is the“image” of the epitope bound by the first monoclonal antibody. Thus, inthis instance, the anti-idiotypic monoclonal antibody could be used forimmunization since the anti-idiotype monoclonal antibody binding domaineffectively acts as an antigen.

[0032] When the monoclonal antibodies of the invention are used in theform of fragments, such as, for example, Fab and F(ab′)₂, and especiallywhen these fragments are therapeutically labeled, any isotype can beused since amelioration of the malignancy in these situations is notdependent upon complement-mediated cytolytic destruction of those cellsbearing the SK1 antigen.

[0033] The monoclonal antibodies of the invention can be used in anyanimal in which it is desirable to administer in vitro or in vivoimmunodiagnosis or immunotherapy. The term “animal” as used herein ismeant to include both humans as well as non-humans.

[0034] The term “antibody” as used in this invention is meant to includeintact molecules as well as fragments thereof, such as for example, Faband F(ab′)₂, which are capable of binding the epitopic determinant.

[0035] The invention provides polynucleotides encoding the SK1 antigen.These polynucleotides include DNA, cDNA and RNA sequences which encodeSK1. It is understood that all polynucleotides encoding all or a portionof SK1 are also included herein, as long as they encode a polypeptidewith SK1 biological activity. Such polynucleotides include naturallyoccurring, synthetic, and intentionally manipulated polynucleotides. Forexample, SK1 polynucleotide may be subjected to site-directedmutagenesis. The polynucleotide sequence for SK1 also includes antisensesequences. The polynucleotides of the invention include sequences thatare degenerate as a result of the genetic code. There are 20 naturalamino acids, most of which are specified by more than one codon.Therefore, all degenerate nucleotide sequences are included in theinvention as long as the amino acid sequence of SK1 polypeptide encodedby the nucleotide sequence is functionally unchanged.

[0036] Minor modifications of the recombinant SK1 primary amino acidsequence may result in proteins which have substantially equivalentactivity and antigenicity as compared to the SK1 polypeptide describedherein. Such modifications may be deliberate, as by site-directedmutagenesis, or may be spontaneous. All of the polypeptides produced bythese modifications are included herein as long as the biologicalactivity and function of SK1 still exists. Further, deletion of one ormore amino acids can also result in a modification of the structure ofthe resultant molecule without significantly altering its biologicalactivity. This can lead to the development of a smaller active moleculewhich would have broader utility. For example, one can remove amino orcarboxy terminal amino acids which are not required for SK1 biologicalactivity.

[0037] The nucleotide sequence encoding the SK1 polypeptide of theinvention includes the disclosed sequence and conservative variationsthereof. The term “conservative variation” as used herein denotes thereplacement of an amino acid residue by another, biologically similarresidue. Examples of conservative variations include the substitution ofone hydrophobic residue such as isoleucine, valine, leucine ormethionine for another, or the substitution of one polar residue foranother, such as the substitution of arginine for lysine, glutamic foraspartic acids, or glutamine for asparagine, and the like. The term“conservative variation” also includes the use of a substituted aminoacid in place of an unsubstituted parent amino acid provided thatantibodies raised to the substituted polypeptide also immunoreact withthe unsubstituted polypeptide.

[0038] DNA sequences of the invention can be obtained by severalmethods. For example, the DNA can be isolated using hybridizationtechniques which are well known in the art. These include, but are notlimited to: 1) hybridization of genomic or cDNA libraries with probes todetect homologous nucleotide sequences and 2) antibody screening ofexpression libraries to detect cloned DNA fragments with sharedstructural features.

[0039] Preferably the SK1 polynucleotide of the invention is derivedfrom a mammalian organism, and most preferably from a mouse, rat, orhuman. Screening procedures which rely on nucleic acid hybridizationmake it possible to isolate any gene sequence from any organism,provided the appropriate probe is available. Oligonucleotide probes,which correspond to a part of the sequence encoding the protein inquestion, can be synthesized chemically. This requires that short,oligopeptide stretches of amino acid sequence must be known. The DNAsequence encoding the protein can be deduced from the genetic code,however, the degeneracy of the code must be taken into account. It ispossible to perform a mixed addition reaction when the sequence isdegenerate. This includes a heterogeneous mixture of denatureddouble-stranded DNA. For such screening, hybridization is preferablyperformed on either single-stranded DNA or denatured double-strandedDNA. Hybridization is particularly useful in the detection of cDNAclones derived from sources where an extremely low amount of mRNAsequences relating to the polypeptide of interest are present. In otherwords, by using stringent hybridization conditions directed to avoidnon-specific binding, it is possible, for example, to allow theautoradiographic visualization of a specific cDNA clone by thehybridization of the target DNA to that single probe in the mixturewhich is its complete complement (Wallace, et al., Nucl. Acid Res.,9:879, 1981).

[0040] The development of specific DNA sequences encoding SK1 can alsobe obtained by: 1) isolation of double-stranded DNA sequences from thegenomic DNA; 2) chemical manufacture of a DNA sequence to provide thenecessary codons for the polypeptide of interest; and 3) in vitrosynthesis of a double-stranded DNA sequence by reverse transcription ofmRNA isolated from a eukaryotic donor cell. In the latter case, adouble-stranded DNA complement of mRNA is eventually formed which isgenerally referred to as cDNA.

[0041] Of the three above-noted methods for developing specific DNAsequences for use in recombinant procedures, the isolation of genomicDNA isolates is the least common. This is especially true when it isdesirable to obtain the microbial expression of mammalian polypeptidesdue to the presence of introns.

[0042] The synthesis of DNA sequences is frequently the method of choicewhen the entire sequence of amino acid residues of the desiredpolypeptide product is known. When the entire sequence of amino acidresidues of the desired polypeptde is not known, the direct synthesis ofDNA sequences is not possible and the method of choice is the synthesisof cDNA sequences. Among the standard procedures for isolating cDNAsequences of interest is the formation of plasmid- or phage-carryingcDNA libraries which are derived from reverse transcription of mRNAwhich is abundant in donor cells that have a high level of geneticexpression. When used in combination with polymerase chain reactiontechnology, even rare expression products can be cloned. In those caseswhere significant portions of the amino acid sequence of the polypeptideare known, the production of labeled single or double-stranded DNA orRNA probe sequences duplicating a sequence putatively present in thetarget cDNA may be employed in DNA/DNA hybridization procedures whichare carried out on cloned copies of the cDNA which have been denaturedinto a single-stranded form (Jay, et al., Nucl. Acid Res., 11:2325,1983).

[0043] A cDNA expression library, such as lambda gt11, can be screenedindirectly for SK1 peptides having at least one epitope, usingantibodies specific for SK1. Such antibodies can be either polyclonallyor monoclonally derived and used to detect expression product indicativeof the presence of SK1 cDNA.

[0044] DNA sequences encoding SK1 can be expressed in vitro by DNAtransfer into a suitable host cell. “Host cells” are cells in which avector can be propagated and its DNA expressed. The term also includesany progeny of the subject host cell. It is understood that all progenymay not be identical to the parental cell since there may be mutationsthat occur during replication. However, such progeny are included whenthe term “host cell” is used. Methods of stable transfer, meaning thatthe foreign DNA is continuously maintained in the host, are known in theart.

[0045] In the present invention, the SK1 polynucleotide sequences may beinserted into a recombinant expression vector. The term “recombinantexpression vector” refers to a plasmid, virus or other vehicle known inthe art that has been manipulated by insertion or incorporation of theSK1 genetic sequences. Such expression vectors contain a promotersequence which facilitates the efficient transcription of the insertedgenetic sequence of the host. The expression vector typically containsan origin of replication, a promoter, as well as specific genes whichallow phenotypic selection of the transformed cells. Vectors suitablefor use in the present invention include, but are not limited to theT7-based expression vector for expression in bacteria (Rosenberg, etal., Gene 56:125, 1987), the pMSXND expression vector for expression inmammalian cells (Lee and Nathans, J. Biol. Chem., 263:3521, 1988) andbaculovirus-derived vectors for expression in insect cells. The DNAsegment can be present in the vector operably linked to regulatoryelements, for example, a promoter (e.g., T7, metallothionein I, orpolyhedrin promoters).

[0046] Polynucleotide sequences encoding SK1 can be expressed in eitherprokaryotes or eukaryotes. Hosts can include microbial, yeast, insectand mammalian organisms. Methods of expressing DNA sequences havingeukaryotic or viral sequences in prokaryotes are well known in the art.Biologically functional viral and plasmid DNA vectors capable ofexpression and replication in a host are known in the art. Such vectorsare used to incorporate DNA sequences of the invention.

[0047] Transformation of a host cell with recombinant DNA may be carriedout by conventional techniques as are well known to those skilled in theart. Where the host is prokaryotic, such as E. coli, competent cellswhich are capable of DNA uptake can be prepared from cells harvestedafter exponential growth phase and subsequently treated by the CaCl₂method using procedures well known in the art. Alternatively, MgCl₂ orRbCl can be used. Transformation can also be performed after forming aprotoplast of the host cell if desired.

[0048] When the host is a eukaryote, such methods of transfection of DNAas calcium phosphate co-precipitates, conventional mechanical proceduressuch as microinjection, electroporation, insertion of a plasmid encasedin liposomes, or virus vectors may be used. Eukaryotic cells can also becotransformed with DNA sequences encoding the SK1 of the invention, anda second foreign DNA molecule encoding a selectable phenotype, such asthe herpes simplex thymidine kinase gene. Another method is to use aeukaryotic viral vector, such as simian virus 40 (SV40) or bovinepapilloma virus, to transiently infect or transform eukaryotic cells andexpress the protein. (see for example, Eukaryotic Viral Vectors, ColdSpring Harbor Laboratory, Gluzman ed., 1982).

[0049] Isolation and purification of microbial expressed polypeptide, orfragments thereof, provided by the invention, may be carried out byconventional means including preparative chromatography andimmunological separations involving monoclonal or polyclonal antibodies.

DIAGNOSTIC USES

[0050] The monoclonal antibodies of the invention are suited for use,for example, in immunoassays in which they can be utilized in liquidphase or bound to a solid phase carrier. In addition, the monoclonalantibodies in these immunoassays can be detectably labeled in variousways. Examples of types of immunoassays which can utilize monoclonalantibodies of the invention are competitive and non-competitiveimmunoassays in either a direct or indirect format. Examples of suchimmunoassays are the radioimmunoassay (RIA) and the sandwich(immunometric) assay. Detection of the antigens using the monoclonalantibodies of the invention can be done utilizing immunoassays which arerun in either the forward, reverse, or simultaneous modes, includingimmunohistochemical assays on physiological samples. Those of skill inthe art will know, or can readily discern, other immunoassay formatswithout undue experimentation.

[0051] The monoclonal antibodies of the invention can be bound to manydifferent carriers and used to detect the presence of SK1. Examples ofwell-known carriers include glass, polystyrene, polypropylene,polyethylene, dextran, nylon, amylases, natural and modified celluloses,polyacrylamides, agaroses and magnetite. The nature of the carrier canbe either soluble or insoluble for purposes of the invention. Thoseskilled in the art will know of other suitable carriers for bindingmonoclonal antibodies, or will be able to ascertain such, using routineexperimentation.

[0052] There are many different labels and methods of labeling known tothose of ordinary skill in the art. Examples of the types of labelswhich can be used in the present invention include enzymes,radioisotopes, fluorescent compounds, colloidal metals, chemiluminescentcompounds, and bioluminescent compounds. Those of ordinary skill in theart will know of other suitable labels for binding to the monoclonalantibody, or will be able to ascertain such, using routineexperimentation. Furthermore, the binding of these labels to themonoclonal antibody of the invention can be done using standardtechniques common to those of ordinary skill in the art.

[0053] For purposes of the invention, SK1 may be detected by themonoclonal antibodies of the invention when present in biological fluidsand tissues. Any sample containing a detectable amount of SK1 can beused. A sample can be a liquid such as urine, saliva, cerebrospinalfluid, blood, serum and the like, or a solid or semi-solid such astissues, feces, and the like, or, alternatively, a solid tissue such asthose commonly used in histological diagnosis.

[0054] Another technique which may also result in greater sensitivityconsists of coupling the antibodies to low molecular weight haptens.These haptens can then be specifically detected by means of a secondreaction. For example, it is common to use such haptens as biotin, whichreacts with avidin, or dinitrophenyl, pyridoxal, and fluorescein, whichcan react with specific anti-hapten antibodies.

[0055] As used in this invention, the term “epitope” is meant to includeany determinant capable of specific interaction with the monoclonalantibodies of the invention. Epitopic determinants usually consist ofchemically active surface groupings of molecules such as amino acids orsugar side chains and usually have specific three dimensional structuralcharacteristics, as well as specific charge characteristics.

[0056] In using the monoclonal antibodies of the invention for the invivo detection of antigen, the detectably labeled monoclonal antibody isgiven in a dose which is diagnostically effective. The term“diagnostically effective” means that the amount of detectably labeledmonoclonal antibody is administered in sufficient quantity to enabledetection of the site having the SK1 antigen for which the monoclonalantibodies are specific.

[0057] The concentration of detectably labeled monoclonal antibody whichis administered should be sufficient such that the binding to thosecells having SK1 is detectable compared to the background. Further, itis desirable that the detectably labeled monoclonal antibody be rapidlycleared from the circulatory system in order to give the besttarget-to-background signal ratio.

[0058] As a rule, the dosage of detectably labeled monoclonal antibodyfor in vivo diagnosis will vary depending on such factors as age, sex,and extent of disease of the individual. The dosage of monoclonalantibody can vary from about 0.01 mg/m² to about 500 mg/m², preferably0.1 mg/m² to about 200 mg/m², most preferably about 0.1 mg/m² to about10 mg/m². Such dosages may vary, for example, depending on whethermultiple injections are given, tumor burden, and other factors known tothose of skill in the art. For in vivo diagnostic imaging, the type ofdetection instrument available is a major factor in selecting a givenradioisotope. The radioisotope chosen must have a type of decay which isdetectable for a given type of instrument. Still another importantfactor in selecting a radioisotope for in vivo diagnosis is that thehalf-life of the radioisotope be long enough so that it is stilldetectable at the time of maximum uptake by the target, but short enoughso that deleterious radiation with respect to the host is minimized.Ideally, a radioisotope used for in vivo imaging will lack a particleemission, but produce a large number of photons in the 140-250 keVrange, which may be readily detected by conventional gamma cameras.

[0059] For in vivo diagnosis radioisotopes may be bound toimmunoglobulin either directly or indirectly by using an intermediatefunctional group. Intermediate functional groups which often are used tobind radioisotopes which exist as metallic ions to immunoglobulins arethe bifunctional chelating agents such as diethylenetriaminepentaceticacid (DTPA) and ethylenediaminetetraacetic acid (EDTA) and similarmolecules. Typical examples of metallic ions which can be bound to themonoclonal antibodies of the invention are ¹¹¹In, ⁹⁷Ru, ⁶⁷Ga, ⁶⁸Ga,⁷²As, ⁸⁹Zr, ⁹⁰Y, and ²⁰¹TI.

[0060] The monoclonal antibodies of the invention can also be labeledwith a paramagnetic isotope for purposes of in vivo diagnosis, as inmagnetic resonance imaging (MRI) or electron spin resonance (ESR). Ingeneral, any conventional method for visualizing diagnostic imaging canbe utilized. Usually gamma and positron emitting radioisotopes are usedfor camera imaging and paramagnetic isotopes for MRI. Elements which areparticularly useful in such techniques include ¹⁵⁷Gd, ⁵⁵Mn, ¹⁶²Dy, ⁵²Cr,and ⁵⁶Fe.

[0061] The monoclonal antibodies of the invention can be used to monitorthe course of amelioration of malignancy in an animal. Thus, bymeasuring the increase or decrease in the number of cells expressing SK1or changes in the concentration of SK1 present in various body fluids,it would be possible to determine whether a particular therapeuticregimen aimed at ameliorating the malignancy is effective.

THERAPEUTIC USES

[0062] The term “ameliorate” denotes a lessening of the detrimentalaffect of the malignancy in the animal receiving therapy. The term“therapeutically effective” means that the amount of monoclonal antibodyor SK1 used is of sufficient quantity to ameliorate the malignancy.

[0063] The term “immunogenically effective amount,” as used in theinvention, is meant to denote that amount of SK1 antigen which isnecessary to induce an ameliorative immune response to the malignancy,for example, by stimulating the production of antibodies which will bindto SK1 epitopes.

[0064] SK1 can be administered parenterally by injection, rapidinfusion, nasopharyngeal absorption, dermal absorption, and orally.Preparations for parenteral administration include sterile or aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Carriers for occlusive dressings can be used to increaseskin permeability and enhance antigen absorption. Liquid dosage formsfor oral administration may generally comprise a liposome solutioncontaining the liquid dosage form. Suitable forms for suspending theliposomes include emulsions, suspensions, solutions, syrups, and elixirscontaining inert diluents commonly used in the art, such as purifiedwater. Besides the inert diluents, such compositions can also includeadjuvants, wetting agents, emulsifying and suspending agents, andsweetening, flavoring, and perfuming agents.

[0065] It is also possible for the antigenic preparations containing SK1to include an adjuvant. Adjuvants are substances that can be used tonon-specifically augment a specific immune response. Normally, theadjuvant and the antigen are mixed prior to presentation to the immunesystem, or presented separately, but into the same site of the animalbeing immunized. Adjuvants can be loosely divided into several groupsbased on their composition. These groups include oil adjuvants (forexample, Freund's Complete and Incomplete), mineral salts (for example,AlK(SO₄)₂, AlNa(SO₄)₂, AlNH₄(SO₄), silica, alum, Al(OH)₃, Ca₃(PO₄)₂,kaolin, and carbon), polynucleotides (for example, poly IC and poly AUacids), and certain natural substances (for example, wax D fromMycobacterium tuberculosis, as well as substances found inCorynebacterium parvum, Bordetella pertussis, and members of the genusBrucella).

[0066] The physical form of the SK1 antigen which is used to immunize ananimal can be either aggregated or non-aggregated. Aggregated SK1 can beproduced from non-aggregated SK1 by such common techniques as, forexample, treatment with glutaraldehyde or other cross-linking agents.The aggregated SK1 thus derived could then be used for purposes ofproducing a malignancy ameliorating composition effective in inducing anactive immune reaction.

[0067] However, regardless of whether an animal is immunized withaggregated or non-aggregated, both of these forms of SK1 should causethe production of antibodies to SK1. Thus, it is possible to use theseanti-SK1 antibodies diagnostically as, for example, in a kit to detectthe presence of SK1 in a specimen.

[0068] The SK1 antigen preparations of the invention can be used toinduce the production of antibodies which will bind to epitopicdeterminants of SK1. A particularly useful method in enhancing theproduction of antibodies to SK1 is to first immunize with the SK1antigenic preparation of the invention followed by a later immunization.

[0069] Many different techniques exist for the timing of theimmunizations when a multiple immunization regimen is utilized. It ispossible to use the antigenic preparation of the invention more thanonce to increase the levels and diversity of expression of theimmunoglobulin repertoire expressed by the immunized animal. Typically,if multiple immunizations are given, they will be spaced one to twomonths apart.

[0070] Generally, the dosage of SK1 administered to an animal will varydepending on such factors as age, condition, sex and extent of disease,if any, and other variables which can be adjusted by one of ordinaryskill in the art.

[0071] The antigenic SK1 preparations of the invention can beadministered as either single or multiple dosages and can vary fromabout 50 mg to about 500 mg for the SK1 antigen per dose, morepreferably about 50 mg to about 300 mg SK1 antigen per dose, mostpreferably about 100 mg to about 200 mg SK1 antigen per dose.

[0072] The monoclonal antibodies of the invention can also be used,alone or in combination with effector cells (Douillard, et al.,Hybridoma, 5 (Supp. 1: S139, 1986), for immunotherapy in an animalhaving a tumor which expresses SK1 antigen with epitopes reactive withthe monoclonal antibodies of the invention.

[0073] When used for immunotherapy, the monoclonal antibodies of theinvention may be unlabeled or labeled with a therapeutic agent. Theseagents can be coupled either directly or indirectly to the monoclonalantibodies of the invention. One example of indirect coupling is by useof a spacer moiety. These spacer moieties, in turn, can be eitherinsoluble or soluble (Diener, et al., Science, 231:148, 1986) and can beselected to enable drug release from the monoclonal antibody molecule atthe target site. Examples of therapeutic agents which can be coupled tothe monoclonal antibodies of the invention for immunotherapy are drugs,radioisotopes, lectins, and toxins.

[0074] The drugs with which can be conjugated to the monoclonalantibodies of the invention include non-proteinaceous as well asproteinaceous drugs. The terms “non-proteinaceous drugs” encompassescompounds which are classically referred to as drugs, for example,mitomycin C, daunorubicin, and vinblastine.

[0075] The proteinaceous drugs with which the monoclonal antibodies ofthe invention can be labeled include immunomodulators and otherbiological response modifiers. The term “biological response modifiers”is meant to encompass substances which are involved in modifying theimmune response in such manner as to enhance the destruction of theantigen bearing tumor for which the monoclonal antibodies of theinvention are specific. Examples of immune response modifiers includesuch compounds as lymphokines. Lymphokines include tumor necrosisfactor, interleukins 1, 2, and 3, lymphotoxin, macrophage activatingfactor, migration inhibition factor, colony stimulating factor, andinterferon. Interferons with which the monoclonal antibodies of theinvention can be labeled include alpha-interferon, beta-interferon, andgamma-interferon and their subtypes.

[0076] In using radioisotopically conjugated monoclonal antibodies ofthe invention for immunotherapy certain isotypes may be more preferablethan others depending on such factors as leukocyte distribution as wellas isotype stability and emission. If desired, the tumor celldistribution can be evaluated by the in vivo diagnostic techniquesdescribed above. Depending on the malignancy some emitters may bepreferable to others. In general, alpha and beta particle-emittingradioisotopes are preferred in immunotherapy. For example, if an animalhas solid tumor foci, as in a carcinoma, a high energy beta emittercapable of penetrating several millimeters of tissue, such as ⁹⁰Y, maybe preferable. On the other hand, if the malignancy consists of simpletarget cells, as in the case of leukemia, a short range, high energyalpha emitter, such as ²¹²Bi, may be preferable. Examples ofradioisotopes which can be bound to the monoclonal antibodies of theinvention for therapeutic purposes are ¹²⁵I, ¹³¹I, ⁹⁰Y, ⁶⁷Cu, ²¹²Bi,²¹¹At, ²¹²Pb, ⁴⁷Sc, ¹⁰⁹Pd, and ¹⁸⁸Re.

[0077] Lectins are proteins, usually isolated from plant material, whichbind to specific sugar moieties. Many lectins are also able toagglutinate cells and stimulate lymphocytes. However, ricin is a toxiclectin which has been used immunotherapeutically. This is preferablyaccomplished by binding the alpha-peptide chain of ricin, which isresponsible for toxicity, to the antibody molecule to enable sitespecific delivery of the toxic effect.

[0078] Toxins are poisonous substances produced by plants, animals, ormicroorganisms that, in sufficient dose, are often lethal. Diphtheriatoxin is a substance produced by Corynebacterium diphtheria which can beused therapeutically. This toxin consists of an alpha and beta subunitwhich under proper conditions can be separated. The toxic A componentcan be bound to an antibody and used for site specific delivery to a SK1bearing cell for which the monoclonal antibodies of the invention arespecific. Other therapeutic agents which can be coupled to themonoclonal antibodies of the invention are known, or can be easilyascertained, by those of ordinary skill in the art.

[0079] The labeled or unlabeled monoclonal antibodies of the inventioncan also be used in combination with therapeutic agents such as thosedescribed above. Especially preferred are therapeutic combinationscomprising the monoclonal antibody of the invention and immunomodulatorsand other biological response modifiers.

[0080] Thus, for example, the monoclonal antibodies of the invention canbe used in combination with alpha-interferon. This treatment modalityenhances monoclonal antibody targeting of carcinomas by increasing theexpression of monoclonal antibody reactive antigen by the carcinomacells (Greiner, et al., Science, 235:895, 1987). Alternatively, themonoclonal antibody of the invention could be used, for example, incombination with gamma-interferon to thereby activate and increase theexpression of Fc receptors by effector cells which, in turn, results inan enhanced binding of the monoclonal antibody to the effector cell andkilling of target tumor cells. Those of skill in the art will be able toselect from the various biological response modifiers to create adesired effector function which enhances the efficacy of the monoclonalantibody of the invention.

[0081] When the monoclonal antibody of the invention is used incombination with various therapeutic agents, such as those describedherein, the administration of the monoclonal antibody and thetherapeutic agent usually occurs substantially contemporaneously. Theterm “substantially contemporaneously” means that the monoclonalantibody and the therapeutic agent are administered reasonably closetogether with respect to time. Usually, it is preferred to administerthe therapeutic agent before the monoclonal antibody. For example, thetherapeutic agent can be administered 1 to 6 days before the monoclonalantibody. The administration of the therapeutic agent can be daily, orat any other interval, depending upon such factors, for example, as thenature of the tumor, the condition of the patient and half-life of theagent.

[0082] Using the monoclonal antibodies of the invention, it is possibleto design therapies combining all of the characteristics describedherein. For example, in a given situation it may be desirable toadminister a therapeutic agent, or agents, prior to the administrationof the monoclonal antibodies of the invention in combination witheffector cells and the same, or different, therapeutic agent or agents.For example, it may be desirable to treat patients with malignantdisease by first administering gamma-interferon and interleukin-2 dailyfor 3 to 5 days, and on day 5 administer the monoclonal antibody of theinvention in combination with effector cells as well asgamma-interferon, and interleukin-2.

[0083] It is also possible to utilize liposomes with the monoclonalantibodies of the invention in their membrane to specifically deliverthe liposome to the area of the tumor expressing SK1. These liposomescan be produced such that they contain, in addition to the monoclonalantibody, such immunotherapeutic agents as those described above whichwould then be released at the tumor site (Wolff, et al., Biochemical etBiophysical Acta, 802:259, 1984).

[0084] The dosage ranges for the administration of the monoclonalantibodies of the invention are those large enough to produce thedesired effect in which the symptoms of the malignant disease areameliorated. The dosage should not be so large as to cause adverse sideeffects, such as unwanted cross-reactions, anaphylactic reactions, andthe like. Generally, the dosage will vary with the age, condition, sexand extent of the disease in the patient and can be determined by one ofskill in the art. The dosage can be adjusted by the individual physicianin the event of any complication. Dosage can vary from about 0.1 mg/kgto about 2000 mg/kg, preferably about 0.1 mg/kg to about 500 mg/kg, inone or more dose administrations daily, for one or several days.Generally, when the monoclonal antibodies of the invention areadministered conjugated with therapeutic agents lower dosages,comparable to those used for in vivo immunodiagnostic imaging, can beused.

[0085] The monoclonal antibodies of the invention can be administeredparenterally by injection or by gradual perfusion over time. Themonoclonal antibodies of the invention can be administeredintravenously, intraperitoneally, intramuscularly, subcutaneously,intracavity, or transdermally, alone or in combination with effectorcells.

[0086] Preparations for parenteral administration include sterileaqueous or non-aqueous solutions, suspensions, and emulsions. Examplesof non-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's, or fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives may also be present such as,for example, antimicrobials, anti-oxidants, chelating agents, and inertgases and the like.

[0087] The invention also relates to a method for preparing a medicamentor pharmaceutical composition comprising the SK1 antigen, or themonoclonal antibodies of the invention, the medicament being used fortherapy of malignant disorders.

[0088] The above disclosure generally describes the present invention. Amore complete understanding can be obtained by reference to thefollowing specific examples which are provided for purposes ofillustration only, and are not intended to limit the scope of theinvention.

EXAMPLE 1 Preparation of Hybridoma Cell Lines Producing MonoclonalAntibody to SK1

[0089] Lymphocytes isolated from a lymph node of a 60 year-old male withDuke's ‘B’ colon carcinoma were used to generate hybridoma cell linesproducing monoclonal antibody to SK1. Lymphocytes were cultured incomplete medium with either 25% supernatant of pokeweedmitogen-stimulated T lymphocytes (sPWM-T) or 25% supernatant of mixedlymphocyte culture (sMLC) (vol/vol). Carcinoembryonic antigen (CEA) wasused for in vitro immunization (IVI). Purified CEA at variousconcentrations was added to the stimulated lymphocytes for a period of 5to 6 days. At the end of the culture, the viability of the lymphocyteswas determined by 0.05% trypan blue exclusion prior to fusion with humancell line SHFP-1. The mean viability was approximately 70%.

[0090] Briefly, sPWM-T was prepared by first obtaining peripheral bloodlymphocytes by FICOLL-HYPAQUE™ density gradient centrifugation(Pharmacia, Piscataway, N.J.). The lymphocytes were then treated withL-leucine methyl ester, 2.5 mmol/L (Sigma, St. Louis, Mo.), to reduceinhibitory cells (natural killer-like cells, cytotoxic T lymphocytes,and a subset of CD8⁺ T cells) for IVI, as described previously (Thiele,et al., J.Immunol., 134:786, 1985). The T helper-enriched preparationwas obtained by incubating the L-leucine methyl ester-treatedlymphocytes with sheep red blood cells (Cappel, Durham, N.C.). The sheepred blood cells were then lysed with red blood cell lysing solution(Sigma). A 1:100 dilution of pokeweed mitogen (GIBCO, Grand Island,N.Y.) was added to T cells at a concentration of 4×10⁹/L in completeRPMI 1640 supplemented with 10% autologous serum and incubated for 48hours. The supernatant obtained after centrifugation to remove thepokeweed mitogen-stimulated T cells was designated sPWM-T. Supernatantof mixed lymphocyte culture (sMLC) was prepared by mixing heterologouslymphocytes of two patients at a ratio of 1:1, with a finalconcentration of 4×10⁹/L, and cultured for 72 hours. Both supernatantswere stored at −20° C. until used.

[0091] A human B lymphoblastoid cell line derived from parental WIL-2cells (Glassy, J.Tissue Culture Methods, 12:85, 1989), SHFP-1, was thehuman fusion partner cell line. Colon cancer cell lines HT-29 and Caco2were obtained from American Type Culture Collection, Rockville, Md.These cell lines were cultured in RPMI 1640 medium supplemented with 10%to 20% fetal calf serum.

[0092] In the fusion procedure, one part of lymphocytes from the lymphnodes and two parts of SHFP-1 cells were fused with 50% polyethyleneglycol 1500 (Behringer Mannheim, Germany). The hybrids were selected bymedium containing hypoxanthine, aminopterin, and thymidine (BehringerMannheim). Reactivity to HT-29 cells, reactivity to carcinoembryonicantigen, and isotype of immunoglobulin by enzyme-linked immunosorbentassay (ELISA) were determined for the supernatant of each visiblehybridoma. The reactive clones were subcloned by limiting dilution(Glassy, et al., Cancer Invest., 5:449, 1987).

[0093] Immunoglobulin production and tumor reactivity were evaluated byenzyme-linked immunosorbent assay (ELISA). The ELISA plates wereprepared by coating the plates with HT-29 or Caco2 cells at 5×10⁴ cellsper well. After blocking with phosphate-buffered saline-0.5% bovineserum albumin solution, 50 μL of the testing supernatant was added toeach well for 1 to 2 hours at room temperature. After washing,horseradish peroxidase-conjugated goat anti-human immunoglobulin(anti-IgM and anti-IgG were from Jackson, West Grove, Pa., and anti-IgAwas from Zymed, San Francisco, Calif.) was added for an incubation of 45minutes. The substrate, orthophenylenediamine, was added for colorreaction. The plates were read at 492 nm with a microplate reader (MR700, Dynatech Laboratories, Inc., Chantilly, Va.). The backgroundoptical density (OD_(b)) was obtained by substituting phosphate-bufferedsaline-bovine serum albumin solution for the testing supernatant and wasfound to be 0.050 or lower. At the 96-well stage, supernatants with ODsgreater than 0.250 were regarded as positive. Subsequently, because fewIgM-secreting clones bound nonspecifically to the ELISA plates, newcriteria were established to evaluate the ELISA results:

[0094] Criterion A:ΔOD<1.000 and OD_(s)−OD_(f)>0.200, and

[0095] Criterion B:ΔOD>1.000 and OD_(s)−OD_(f)>0.150,

[0096] where ΔOD=OD_(s)−OD_(b), OD_(s) (sample OD) represents the ODobtained from specific binding of supernatant to the antigen-coatedELISA plates, and OD_(f) (false OD) represents the OD obtained fromnonspecific binding of supernatant to the ELISA plates without antigencoating.

EXAMPLE 2 Immunological Reactivity of HuMAb(SK1)

[0097] Various human carcinoma cell lines were tested for theirreactivity with HuMAb(SK1). Cell lines tested were HT29, Caco2, COL205,COL320DM, Panc-1, KATO III, Calu-1, A375, and HTB63 (ATCC, Rockville,Md.) and P3 and M21 (UCLA).

[0098] All the cells were maintained in RPMI 1640 medium (Whittaker,Wallsville, Md.) supplemented with 10-20% FCS (Hyclone Lab., Logan,Utah) and 2 mM L-glutamine (Whittaker) except HT29 cells which weregrown in McCoy's 5A (UCSD Core Facility, La Jolla, Calif.); Panc-1 andCalu-1 cells were grown in DME-high glucose medium (UCSD Core Facility,La Jolla, Calif.). Twenty ml of cell suspension at 5×10⁴/ml were addedto each petri dish containing a sterile glass slide. After variousperiods of culture, the slides were harvested and used for eitherimmunoperoxidase (IP) or immunofluorescence (IF) staining.

[0099] In preparing the slides for staining, the slides with seededcells were washed in PBS and fixed with cold acetone. For indirect IPstaining, fixed cells were incubated with the HuMAb (SK1) supernatant(10 μg/ml) and subsequently developed with horseradishperoxidase-conjugated goat anti-human IgM, followed by diaminobenzidine(DAB; Sigma) containing 0.01% H₂O₂. After counterstaining with Mayer'shematoxylin, the specimens were cleared with ammonium peroxide andmounted with aqua-mount. In IF stainings, a 1:200 dilution of FITCconjugated goat antibody to human IgM (Boehringer Mannheim) was used assecond antibody and reactivities were evaluated by Nikon fluorescencemicroscopy. Controls consisted of secondary antibody and cells.

[0100] In HT29 cells (colon cancer), one day old cultures and peripheraltumor cells of 3 day old colonies were stained intensely. This was incontrast to the infrequent staining observed in the centers of thecolonies. When cells were confluent on the fifth day, the staining ofSK1 was uniformly diminished. The decreased antigen expression as afunction of time was also observed in cultured Calu-1 cells (lungcancer). The antigen was evenly detected in the cytoplasm at thebeginning of the culture (Day 1); subsequently, it concentrated at oneend of the nucleus and the nuclei were often dented toward the antigenconcentrated areas. On Days 4 and 5, the antigen became diffuse and thereactivities were weakened and eventually disappeared by day 7. Asimilar immunocytochemical finding was seen with Panc-1 cells(pancreatic cancer). This data suggests that the expression of SK1 isproliferation related.

EXAMPLE 3 Flow Cytometry

[0101] HT29 cells were examined by flow cytometry to determine membraneand cytoplasmic staining of SK1 using HuMAb(SK1). The HT29 cells wereharvested from 1 day, 2 day, and 5 day old tissue cultures by 0.2%ethylenediamine tetra-acetic acid (EDTA) in Ca²⁺ and Mg²⁺ free PBS.Cells were washed with PBA (PBS with 1% albumin). For cytoplasmicstaining, cells were fixed with 0.5% paraformaldehyde for 30 min andsubsequently incubated with supernatant containing HuMAb(SK1) (10 to 25μg/ml) for 1 h. After washing, a 1:500 dilution of FITC-conjugated goatanti-human IgM (Boehringer Mannheim Biochemicals, Indianapolis, Ind.)was added for 1 h. All steps were performed at 4° C. Five to twentythousand cells were examined using an ORTHO CYTOFLUOROGRAF™ 50-H withargon laser for FITC and PI (488 nm), interfaced with a Data General2150 Computer. The autofluorescence of unstained cells and secondantibody control were included. The non-fixed viable cells were used formembrane reactivity and were fixed with 1% formalin upon the completionof staining. DNA was stained with propidium iodide (PI, Sigma) at 50μg/ml for 30 min in 10 mM Tris, pH 7.0, with 5 mM MgCl₂.

[0102] Flow cytometric studies showed that antigen SK1 was both on thecell surface and in the cytoplasm. A decrease of SK1 positive cells as afunction of in vitro culturing time was observed by both surface andintracellular staining with HuMAb(SK1) (Table 1). TABLE 1 PROGRESSIVELOSS OF AgSK1 IN NONPROLIFERATING CELLS % AgSK1 POSITIVE CELLS Day 1 Day5 Cyto- Day 2 Cyto- Cell Line plasmic Surface Cytoplasmic Surfaceplasmic Surface HT29 89 44 66  6 47 0 Panc1 81 71 65 51 46 0

[0103] In conjunction with DNA analysis of proliferating HT29, the SK1was detected throughout the cell cycle (from G1 to M) suggesting thatSK1 was being synthesized at early G1 phase and persisted thereafter.

[0104] The reactivity of HuMAb(SK1) was also tested on histologicalspecimens derived from various tumor tissues. TABLE 2 REACTIVITY OFHuMAb(SK1) WITH VARIOUS TUMOR TISSUES TUMOR TISSUE REACTIVITY¹colorectal 10/10 gastric 1/1 pancreatic 1/1 lung (adenocarcinoma) 2/2breast 0/2 melanoma 0/4 sarcoma (soft tissue) 0/4

[0105] As shown in Table 2, HuMAb(SK1) was highly reactive with tumorsof the gut, pancreas, and lung, but did not react with specimens ofbreast, melanoma, and soft tissue sarcoma.

EXAMPLE 4 Characterization of SK1

[0106] The effect of various agents on antigen SK1 was evaluated usingELISA plates coated with HT29 cells. The ELISA plates were treated withvarious agents under the following conditions: 0.45 U/ml neuraminidase(Type III, Sigma) pH 5.5, 37° C., overnight; periodic acid (Sigma) 5 mMin sodium acetate buffer, pH 4.5, for 45 min at RT; 0.25% trypsin(Gibco, Grand Island, N.Y.), pH 7.8, 37° C. for 20 min; and heattreatment at 100° C. for 2 min. In determining the sensitivity of SK1 toNP-40, SK1 positive cells were mixed with NP-40 and then centrifuged.SK1 reactivity was then tested on the harvested supernatant using ELISA.The sensitivity of SK1 to trypsin and heat was further analyzed bySDS-PAGE and Western blot. As summarized in Table 3, treating targetcells with neuraminidase, trypsin, periodate, and heat abolished thebinding with HuMAb(SK1) suggesting a sialoglycoprotein nature of SK1.TABLE 3 SENSITIVITY OF AgSK1 TO VARIOUS AGENTS AGENT Periodic Un-Neuraminidase Heat Trypsin Acid NP-40 treated Reaction¹ − − − − + +

[0107] SK1 was also evaluated using lysates of various cell lines. Inpreparing the lysates, HT29, Panc-1, KATO III and Calu-1 cells werewashed 3 times in Tris-buffered saline (TBS) and resuspended with TBS at5×10⁷ cells/ml. The cell lysates were prepared by repeated freezing andthawing (4×) and centrifugation at 3000×g for 10 min. The supernatantswere collected and stored at −70° C. until use. The cell lysates werethen tested by SDS-PAGE and Western Blot.

[0108] Electrophoresis was performed using the Novex X-cell mini-gelsystem (Novex, San Diego, Calif.). Precast 8-16% Tris-glycine gels(Novex) were used to run all samples. Following electrophoresis,proteins were transferred onto nitrocellulose membranes using theBio-Rad transfer system (Richmond, Calif.). Protein standards andprestained standards (Sigma) were used simultaneously. After blockingwith TBA (TBS—1% albumin) the membranes were washed, dried, andincubated in hybridoma-supernatants at 25 μg/ml. Alkaline phosphataseconjugated goat anti-human IgM (American, Qualex, La Marada, Calif.) ata dilution of 1:3000 was used as the secondary antibody. The bands weredeveloped using the Proto Blot System from Promega.

[0109] These studies showed that SK1 is a two chain structure withmolecular weights estimated to be 42-46 kDa by gel electrophoresis andWestern blot under both reducing and non-reducing conditions. Theantigen was identified in cell lysates of 1 to 2 day old cultures of thefollowing cells: HT29, Panc-1, KATO III. The antigen was marginallydetectable when the 5 day cultures were used. In contrast to the abovecarcinoma cells, the melanoma cell lines HTB63, HTB66, HTB71, HTB72,HTB73, UCLA M21, and UCLA M14 had no detectable antigen SK1 by both gelelectrophoresis with Western blot and cytoimmunoperoxidase staining.

EXAMPLE 5 Biological Activity of Antigen SK1

[0110] The following experiments provide both in vitro and in vivo datawhich support the use of monoclonal antibody SK1 in the treatment ofanimals, including humans.

[0111] The Examples above and the following experiments show in in vitrostudies demonstrating the specific reactivity of SK-1 monoclonalantibody of the invention with colon cancer cells. The results are shownin an SK1-monoclonal antibody-mediated complement-dependent cellularcytotoxicity (CDCC) assay against carcinoma cell lines and fresh coloncarcinoma cells. Use of the CDCC assay is one of the primary methodscurrently in use to evaluate an antibody's ability to lyse target cells.

[0112] AgSK1 positive cells, such as the KATO-III cell line were used astargets. An antibody capable of fixing complement (a family of proteinsin serum which are capable of causing the lysis/death of certain celltypes) was incubated with the target cells and then complement. Thetarget antigen positive cells were loaded with 51-Cr and the release ofthe radioactive CPM is directly proportional to the number of cellslysed.

[0113] Approximately 2-5×10⁶ cells were incubated with 200 uCi of 51-Cr,in a total volume of not more than 0.5 ml, at 37° C. for 1 hr, then atroom temperature for 15-20 min to allow for the release of “looselybound” 51-Cr. The cells were then counted, adjusted to a concentrationof 5000 cpm/50 ul/well, and 50 ul added to each well of a 96-well plate.Upon completion, the appropriate concentration of the test monoclonalantibody was added to each well and incubated for 60 min. The plates arecentrifuged (150×g for 5 min), “flicked” off supernatant, then 100 ul offresh media was added to each well, then the wash step was repeated.After flicking off the final wash solution, 50 ul of the test complement(Pel-Freeze; HLA-ABC absorbed) at the appropriate dilution was added tothe wells for a 45 min incubation at 37° C. Then, 50 ul of fresh mediawas added to each well and centrifuged as before. Following thecentrifugation, 50 ul of supernatant was removed from each well andcounted in a gamma counter. Table 4 shows that, as described in thepresent patent application, the SK1 monoclonal antibody selectively andeffectively lysed colon cancer cells (HT29), pancreatic cancer cells(Panc-1) and cancer cells derived from surgical specimens (Colon cancercells pt.#1 and #2) in the CDCC assay.

[0114] In contrast, antibody (10 ug/ml) and complement alone showed nodetectable cytotoxicity, and normal colonic mucosal cells bearing nodetectable antigen was resistant to SK1 in the CDCC assay (Table 4).Specific cytotoxicity was shown to be dose dependent. CDCC was mostremarkable at the highest testing dose, i.e., 10 ug/ml. The specificcytotoxicity of SK1 against colon cancer cells in patient #1 (pt #1) wasdetectable at a serial dilution of antibody up to 1:16. TABLE 4 SK1MEDIATED CDCC AGAINST CARCINOMA CELL LINES AND FRESH COLON CARCINOMACELLS CELLS CDCC % HT29 100 Panc-1 83 Colon Cancer 81 (Pt. #1) Normalcolon mucosal 15 cells (Pt. #1) Colon cancer cells 60 (Pt. #2)

[0115] An additional in vitro CDCC assay was performed as describedabove using Kato-III, Panc-1, or primary colon cells. The results areshown in Table 5. The results of the assay show between 65% and 100%lysis of SK1⁺ cells by SK1 antibody. TABLE 5 CDCC ANALYSIS AgSK1⁺ %Surface¹ % Killing Expt Cell Line % Release AgSK1⁺ AgSK1⁺ Cells 1Kato-III 25.6% 23.2%  90% 2 Kato-III  6.0%  4.0%  65% 3 Panc-1 18.0%21.0% 100% 4 Kato-III 25.7% 28.3% 100% 5 Kato-III  4.3%  6.6% 100% 6Primary colon 20.0% N.D. N.D.

[0116] In addition, in vivo data showing the pharmacological effects ofSK1 in a nude mouse model, is described in Table 6 and FIG. 1. Briefly,Panc-1, pancreatic cancer cells (5×10⁶ cells/mouse), were injected intothree groups of nude mice (Balb/c, Harlan, Sprague-Dawley) on day 0.Immediately after tumor implantation, one group of mice was injectedintraperitoneally (IP) with PBS (500 ul/mouse), one group withpolyclonal human IgM (300 ug/mouse in 500 ul), and one group wasinjected with the SK1 monoclonal antibody of the invention (300 ug/mousein 500 ul). Tumor size was measured every week for 4 weeks.

[0117] The tumor size was significantly suppressed in all of the mice inthe group treated with the SK1 monoclonal antibody, as shown in FIG. 1and Table 6. The animal model used for these studies is a well acceptedmodel which is considered to be predictive of results in humans. TABLE 6SK1 NUDE MOUSE XENOGRAFT MODEL WEEK SAMPLE^(a) 1 2 3 4 PBS  128 ±41^(d) 726 ± 251 6820 ± 687 16210 ± 2353 IgM^(b) ▪ 104 ± 56 693 ± 2757273 ± 1297 16938 ± 2387 SK1^(c) ▴ 68 ± 40 148 ± 69 677 ± 259 3118 ±1016 ^(a)Sample injection volumes of PBS, IgM and SK1 were all at 500ul/mouse. Each group consisted of 7 mice, for a total of 21 mice.^(b)Control polyclonal IgM (Sigma) injected (300 ug) immediately afterimplantation of the Panc-1 tumor cells (5 × 10⁶ cells per mouse). ^(c)-SK1 derived from tissue cultrue supernatant was injected (300 ug)immediatedly after implantation of the Panc-1 tumor cells (5 × 10⁶ cellsper mouse). ^(d)${{{Tumor}\quad {size}} = \frac{\left( {{length} \times {width}} \right)^{2}}{2}};{{7\quad {mice}} = {x \pm y}}$

EXAMPLE 6 Characterization of AGSK1

[0118] Previous studies have identified several forms of AgSK1 in humancolon tumors and in the colon adenocarcinoma cell line HT29 (Chang, etal. Cancer Research, 53: 1122-1127, 1993). These forms include a doubletmigrating at about 42-46 kDa on SDS-PAGE. Our analysis showed theexpression of the 42-46 kDa doublet in various human carcinoma celllines using Western blot analysis with the antibody of the invention.

[0119] Cells were harvested at 50% confluency (or 90% as indicated).Whole cells were pelleted, washed in PBS, repelleted and lysed in ahypotonic solution containing 1.0% NP40. The nuclei were separated fromcytosolic proteins via a brief spin at high speed in a microfuge. Thenuclei were lysed in 10% glycerol/Tris pH 6.8 and DNA sheared byrepeated passage through a 25 gauge needle to reduce viscosity. Theprotein concentration of each fraction was determined by BCA assay(Pierce) and 40 ug of each were loaded on a 8-16% mini PAGE gel (Novex)after mixing 1:1 with 2× non-reducing sample loading buffer (Novex). Theproteins were blotted electrophoretically using a modified method ofTowbin (Proc. Nat. Acad. Sci., 76: 5\4350-4354, 1979) in a Novexapparatus. The blot was processed using the procedure described abovefor plaque lift screening.

[0120]FIG. 2 shows that the doublet was detected in the nuclear pelletsof HT29, KatoIII and Colo205 cells. The glioma line, U87MG, and T-cellline, CEM, were negative for the antigen.

EXAMPLE 7 Reactivity of HuMAb-SK1 with Various Tumor Tissues

[0121] Additional tissues were stained using the immunochemical methodsas described in Example 2. The results in Table 6 show the highreactivity of monoclonal antibody SK1 (MAbSK1) with colorectal cancercells. TABLE 6 IMMUNOCHEMICAL STAINING OF HUMAN TISSUES WITH HuMAb-SK1 #Tissues # Tissues # Tissues Tissues AgSK1+ AgSK1 trace AgSK1− ColorectalCancer 24  0 0 Colorectal mucosa 1 0 1 Small intestine mucosa 0 0 2Stomach cancer 1 0 0 Stomach mucosa 0 0 2 Pancreatic cancer 2 0 0Pancreas 0 2 3 Liver 0 2 3 Lung cancer 2 0 0 Lung 0 0 4 Skin squamouscell 1 0 0 cancer Kidney 0 1 4 Brain 0 0 4 Melanoma 0 0 4 Sarcoma 0 0 4Breast cancer 0 0 3 Prostate 0 0 3 Skin 0 0 3 Esophagus 0 0 3 Stomach 00 3 Bladder 0 0 3 Testis 0 0 3 Ovary 0 0 3 Uterus 0 0 3 Spleen 0 0 3Thyroid 0 0 3 Heart 0 0 3 Bone Marrow 0 0 3 Eye 0 0 3 Nerve 0 0 4Skeletal Muscle 0 0 3 Salivary Gland 0 0 1

DEPOSIT OF MATERIALS

[0122] The following cell line has been deposited with the American TypeCulture Collection, 1301 Parklawn Drive, Rockville, Md., USA (ATCC) onOct. 24, 1991: Cell Line ATCC Accession No. SK1 HB 10905

[0123] The deposit was made under the provisions of the Budapest Treatyon the International Recognition of the Deposit of Microorganisms forthe Purpose of Patent Procedure and the Regulations thereunder (BudapestTreaty). This assures maintenance of viable cultures for 30 years fromthe date of deposit. The organisms will be made available by ATCC underthe terms of the Budapest Treaty which assures permanent andunrestricted availability of the progeny of the culture to the publicupon issuance of the pertinent U.S. patent or upon laying open to thepublic of any U.S. or foreign patent application, whichever comes first,and assures availability of the progeny to one determined by the U.S.Commissioner of Patents and Trademarks to be entitled thereto accordingto 35 USC §122 and the Commissioner's rules pursuant thereto (including37 CFR §1.14 with particular reference to 886 OG 638).

[0124] If the culture deposit should die or be lost or destroyed whencultivated under suitable conditions, it will be promptly replaced onnotification with a viable specimen of the same culture. Availability ofa deposited strain is not to be construed as a license to practice theinvention in contravention of the rights granted under the authority ofany government in accordance with its patent laws.

[0125] The invention now being fully described, it will be apparent toone of ordinary skill in the art that many changes and modifications canbe made without departing from the spirit or scope of the invention.

1. A continuous hybridoma cell line which secretes monoclonal antibodieswhich specifically bind to carcinoma specific antigen SK1.
 2. Thehybridoma of claim 1, wherein the hybridoma is ATCC HB
 10905. 3. Amonoclonal antibody which specifically binds to SK1.
 4. The monoclonalantibody of claim 3, which is human.
 5. The monoclonal antibody of claim4, having the specificity of a monoclonal antibody produced by hybridomacell line ATCC HB
 10905. 6. The monoclonal antibody of claim 4, whereinthe monoclonal antibody is produced by hybridoma cell line ATCC HB10905.
 7. An anti-idiotypic antibody to the antibody of claim
 3. 8. Amethod of detecting SK1 which comprises contacting a source suspected ofcontaining SK1 with a diagnostically effective amount of detectablelabeled antibody or fragment thereof, wherein the antibody specificallybinds to SK1 and determining whether the antibody binds to the source.9. The method of claim 8, wherein the antibody is produced by hybridomacell line ATCC HB
 10905. 10. The method of claim 8, wherein thedetecting is in vivo.
 11. The method of claim 10, wherein the detectablelabel is selected from the group consisting of a radioisotope and aparamagnetic label.
 12. The method of claim 8, wherein the detecting isin vitro.
 13. The method of claim 12, wherein the detectable label isselected from the group consisting of a radioisotope, a fluorescentcompound, a colloidal metal, a chemiluminescent compound, abioluminescent compound and an enzyme.
 14. A method of amelioratingmalignant disease in an animal which comprises administering to theanimal a therapeutically effective amount of a monoclonal antibody orfragment thereof, wherein said antibody specifically binds to SK1. 15.The method of claim 14, wherein the malignant disease is a carcinoma.16. The method of claim 14, wherein the antibody is human.
 17. Themethod of claim 14, wherein the monoclonal antibody has the specificityof the monoclonal antibody produced by hybridoma cell line ATCC HB10905.
 18. The method of claim 14, wherein the monoclonal antibody isproduced by hybridoma cell line ATCC HB
 10905. 19. The method of claim14, wherein the administration is parenteral.
 20. The method of claim18, wherein the parenteral administration is by subcutaneous,intramuscular, intraperitoneal, intracavity, transdermal, or intravenousinjection.
 21. The method of claim 14, wherein the administration is ata dosage of about 0.01 mg/kg to about 2000 mg/kg/dose.
 22. The method ofclaim 14, wherein the antibody is administered in combination witheffector cells.
 23. The method of claim 14, wherein the monoclonalantibody is therapeutically labeled.
 24. The method of claim 23, whereinthe therapeutic label is selected from the group consisting of aradioisotope, a drug, an immunomodulator, a biological responsemodifier, a lectin, and a toxin.
 25. The method as in any of claims 14,22 and 23, wherein the antibody is administered substantiallycontemporaneously in combination with a therapeutic agent.
 26. Themethod of claim 25, wherein the therapeutic agent is selected from thegroup consisting of a radioisotope, a drug, an immunomodulator, abiological response modifier, a lectin, and a toxin.
 27. Antigen SK1 insubstantially pure form and mixtures and salts thereof.
 28. The SK1 ofclaim 27 having a molecular weight of 42-46 kD as determined by sodiumdodecyl sulfate polyacrylamide gel eletrophoresis under reducingconditions.
 29. The SK1 of claim 27 comprising an epitope which isspecifically bound by a monoclonal antibody having the specificity of amonoclonal antibody produced by hybridoma cell line ATCC HB
 10905. 30. Amethod of ameliorating malignant disease in an animal which comprisesimmunizing the animal with an immunogenically effective amount of Sk1.31. A method of ameliorating malignant disease in an animal whichcomprises immunizing the animal with an immunogenically effective amountof the anti-idiotypic antibody of claim
 7. 32. A pharmaceuticalcomposition which comprises a therapeutically effective amount of themonoclonal antibodies as in any of claims 3-7, together with apharmacological carrier.
 33. A pharmaceutical composition comprising animmunogenically effective amount of SK1, together with a pharmacologicalcarrier.